International Journal of

Molecular Sciences

Article

Neuroprotective and Anti-Apoptotic Effects ofCSP-1103 in Primary Cortical Neurons Exposed toOxygen and Glucose Deprivation

Vanessa Porrini 1,*, Ilenia Sarnico 1, Marina Benarese 1, Caterina Branca 1, Mariana Mota 1,Annamaria Lanzillotta 1, Arianna Bellucci 1, Edoardo Parrella 1, Lara Faggi 1, Pierfranco Spano 1,2,Bruno Pietro Imbimbo 3 and Marina Pizzi 1,2,*

1 Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia,Italy; ilesar@libero.it (I.S.); marina.benarese@unibs.it (M.B.); c.branca@unibs.it (C.B.);m.coelhodamota@unibs.it (M.M.); annamaria.lanzillotta@unibs.it (A.L.); arianna.bellucci@unibs.it (A.B.);edoardo.parrella@unibs.it (E.P.); lara.faggi@unibs.it (L.F.); pierfranco.spano@unibs.it (P.S.)

2 Department of Neurorehabilitation, IRCCS San Camillo, Via Alberoni 70, 30126 Venice, Italy3 Research & Development, Chiesi Farmaceutici, Via Palermo 26/A, 43100 Parma, Italy;

b.imbimbo@chiesi.com* Correspondence: v.porrini@unibs.it (V.P.); marina.pizzi@unibs.it (M.P.);

Tel.: +39-030-3717502 (V.P.); +39-030-3717501 (M.P.)

Academic Editor: Katalin Prokai-TatraiReceived: 11 October 2016; Accepted: 12 January 2017; Published: 18 January 2017

Abstract: CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduceamyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer’sdisease. Moreover, it was found to improve cognition and reduce brain inflammation in patientswith mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a singlemolecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptionalregulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptoticand neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein theprimary mouse cortical neurons were exposed to oxygen-glucose deprivation (OGD). When addedafter OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 andnuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in amodel of post-ischemic brain injury and provide further mechanistic insights as regards its abilityto reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, thesefindings suggest a potential use of CSP-1103 for the treatment of brain ischemia.

Keywords: CSP-1103; CHF5074; oxygen-glucose deprivation; ischemia; caspase-3; nuclear factor κB(NF-κB); p38; amyloid precursor protein intracellular domain (AICD)

1. Introduction

Stroke is one of the major causes of mortality and disability worldwide [1]. Presently, thereare no clinically effective therapies for stroke recovery, and current treatments offer only limitedbenefits, making ever more urgent the need to develop new therapies. Apoptosis and necrosis are keymechanisms that lead to cell death after cerebral ischemia. In the ischemic core in particular, most of thecells die of necrosis, while apoptosis is mainly involved with the penumbra, the border of the ischemicarea, where the levels of energy and oxygen are sufficient to support apoptotic processes [2–4]. In fact,signs of apoptosis exist, including cytochrome c release, activation of caspases-3 and -9, and terminal

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deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity, thus making this cascade apotential target for neuroprotection [5–8].

CSP-1103 (1-(3′,4′-dichloro-2-fluoro (1,1′-biphenyl)-4-yl)-cyclopropanecarboxylic acid) is an orallybioavailable, brain penetrating, non-steroidal anti-inflammatory drug (NSAID) derivative withmarkedly reduced cyclooxygenase (COX) inhibitory activity [9], in contrast to classic NSAIDs such asibuprofen, in development for the treatment of Alzheimer’s disease (AD) and other neurodegenerativedisorders by CereSpir Incorporated.

CSP-1103 was originally found to limit the β-amyloid (Aβ) plaque burden and amelioratecognitive deficits when chronically administered to transgenic mouse models of AD [9–12]. Based onthe results of preclinical and clinical investigations [13–15], CSP-1103 has been hypothesized to reducethe production and release of pro-inflammatory cytokines by microglia and to enhance microglialphagocytic capacity via an effect on astrocyte-microglia cross-talk. Specifically, CSP-1103 is thoughtto reduce the availability of the astrocytic-signaling molecule soluble CD40 ligand (sCD40L), whichnormally binds the microglial receptor CD40 on the cell surface. The CD40 ligation is a recognizedmechanism driving the pro-inflammatory phenotype of microglia, including the release of cytokinessuch as tumor necrosis factor α (TNF-α), triggering the free radical-mediated tissue damage andlimiting the microglial phagocytic activity [16]. Preclinical evidence shows that CSP-1103 reduces thelevels of a number of pro-inflammatory microglial markers, including inducible nitric oxide synthase(iNOS), interleukin 1 β (IL-1β), and TNF-α, while stimulating the transcription of proteins involved inphagocytosis, such as triggering receptor on myeloid cells 2 (TREM2) [14]. A Phase 1 2-week study inhealthy volunteers and a Phase 2a 12-week study in patients with mild cognitive impairment confirmedin humans the TNF-α inhibiting properties of CSP-1103 and showed a corresponding effect on sCD40Llevels in the cerebrospinal fluid (CSF) [13,15]. Lower CSF total tau levels were also noted [17], whileno effects were seen on soluble Aβ species (Aβ40 and Aβ42) [13,15].

The observation that CSP-1103 binds the amyloid precursor protein intracellular domain (AICD)with sub-micromolar affinity and inhibits AICD nuclear translocation and interaction with targetgene promoters [18], provides a plausible mechanism to explain the various beneficial effects of thecompound through a single molecular target. AICD is a transcriptional regulator that affects theproduction of proteins involved in inflammation, intracellular trafficking, and apoptosis [19]. Amongthe AICD target genes, CSP-1103 was shown to impair the expression of pro-apoptotic tetraspaninKAI1/CD82 in Tg2576 mice [18]. Moreover, “pretreatment” with CSP-1103 was found to reduceapoptosis in diverse experimental settings, including in the SH-SY5Y neuroblastoma cells exposed toAβ25-35 or Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) [20] or the primaryhippocampal neurons exposed to oxygen glucose deprivation (OGD) [21].

These findings prompted us to expand on the anti-apoptotic effects of CSP-1103 by investigatingits activity in a diverse experimental settings mimicking the therapeutic scheme for the “post-ischemic”injury. By using primary cortical neurons exposed to OGD as a cell-based model of cortical brainischemia, we tested the neuroprotective and anti-apoptotic effects of CSP-1103, when added in thepost-OGD period, and the signaling cascade involved. The activity of CSP-1103 was compared to thatof ibuprofen, a classical NSAID with potent COX inhibitory activity.

2. Results

2.1. CSP-1103, but Not Ibuprofen, Reduces Necrosis Induced by OGD in Primary Cortical Neurons

Following OGD, primary cultures of mouse cortical neurons have been shown to undergoapoptosis prior to necrosis. Neuronal cells display TUNEL-positivity and release of cytochromec in the cytosol within 6 h after the OGD, in the absence of lactate dehydrogenase (LDH) release.A secondary necrosis was revealed by the progressive release of cellular LDH that became clearlydetectable in the culture medium 24 h after the OGD [22,23]. The effects of CSP-1103 and ibuprofen

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on neuronal injury were firstly evaluated at the end of the 24 h recovery period by measuring LDHrelease (Figure 1).

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Figure 1. Neuroprotective effect of CSP-1103, but not ibuprofen, in cortical neurons exposed to oxygen glucose deprivation (OGD). (A) Cortical neurons were exposed to 3 h OGD and neuronal death was assessed after 24 h recovery by the lactate dehydrogenase (LDH) assay. CSP-1103, added after the OGD period, increased the survival of injured neurons at different concentrations (1 and 3 μM); (B) Lack of neuroprotective effect of ibuprofen in cortical neurons exposed to 3 h OGD. Different doses of ibuprofen were added after OGD and the neuronal death was measured by the LDH assay 24 h later. Values are expressed as percentage of the LDH released by the cells exposed to OGD. * p < 0.05, ** p < 0.01 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

Cortical neurons were exposed to CSP-1103 or ibuprofen in the post-OGD period, a condition which mimics in vitro the therapeutic scheme for in vivo brain ischemia. CSP-1103, tested at concentrations ranging from 0.5 μM to 30 μM, displayed maximal neuroprotection at 3 μM (Figure 1A). Conversely, no protection was elicited by ibuprofen at any concentration tested in the range 30–1000 μM (Figure 1B). No toxic effects were elicited by CSP-1103 and ibuprofen when added to naïve neuronal cells at the same concentration range (Figure S1).

2.2. CSP-1103, but Not Ibuprofen, Prevents Caspase-3 Activation Induced by OGD in Primary Cortical Neurons

To examine the apoptotic cascade induced by OGD, we measured the activity of caspase-3, a member of the cysteine-dependent, aspartate-specific, proteolytic enzymes family that plays a central role in the propagation of the apoptotic processes. We checked the level of cleaved caspase-3 protein (c-casp-3) in cytosolic extracts of neurons exposed to OGD and treated with the vehicle or the drugs for 6 h in the post-OGD period. Data analysis from western blot (WB) revealed that CSP-1103 at 3 μM significantly reduced caspase-3 cleavage (Figure 2A,B). Conversely, ibuprofen did not modify caspase-3 cleavage, in line with the lack of neuroprotective activity of the drug shown by the LDH release assay (Figure 2A,B).

Furthermore, we checked the immunoreactivity to c-casp-3 in neurons exposed to CSP-1103 (0.5–3 μM) for 24 h in the post-OGD period. Cells were processed for immunocytochemistry, using an antibody specific for c-casp-3 and counterstained with hematoxylin. The data showed increased immunoreactivity for c-casp-3 in cells exposed to OGD. The immunoreactivity was reduced in neurons treated with CSP-1103 at concentrations ≥0.5 μM (Figure 2C–H).

Figure 1. Neuroprotective effect of CSP-1103, but not ibuprofen, in cortical neurons exposed to oxygenglucose deprivation (OGD). (A) Cortical neurons were exposed to 3 h OGD and neuronal death wasassessed after 24 h recovery by the lactate dehydrogenase (LDH) assay. CSP-1103, added after the OGDperiod, increased the survival of injured neurons at different concentrations (1 and 3 µM); (B) Lack ofneuroprotective effect of ibuprofen in cortical neurons exposed to 3 h OGD. Different doses of ibuprofenwere added after OGD and the neuronal death was measured by the LDH assay 24 h later. Values areexpressed as percentage of the LDH released by the cells exposed to OGD. * p < 0.05, ** p < 0.01 vs.OGD value. +, presence of OGD; −, absence of OGD or treatment.

Cortical neurons were exposed to CSP-1103 or ibuprofen in the post-OGD period, a conditionwhich mimics in vitro the therapeutic scheme for in vivo brain ischemia. CSP-1103, tested atconcentrations ranging from 0.5 µM to 30 µM, displayed maximal neuroprotection at 3 µM (Figure 1A).Conversely, no protection was elicited by ibuprofen at any concentration tested in the range 30–1000 µM(Figure 1B). No toxic effects were elicited by CSP-1103 and ibuprofen when added to naïve neuronalcells at the same concentration range (Figure S1).

2.2. CSP-1103, but Not Ibuprofen, Prevents Caspase-3 Activation Induced by OGD in Primary Cortical Neurons

To examine the apoptotic cascade induced by OGD, we measured the activity of caspase-3,a member of the cysteine-dependent, aspartate-specific, proteolytic enzymes family that plays a centralrole in the propagation of the apoptotic processes. We checked the level of cleaved caspase-3 protein(c-casp-3) in cytosolic extracts of neurons exposed to OGD and treated with the vehicle or the drugsfor 6 h in the post-OGD period. Data analysis from western blot (WB) revealed that CSP-1103 at3 µM significantly reduced caspase-3 cleavage (Figure 2A,B). Conversely, ibuprofen did not modifycaspase-3 cleavage, in line with the lack of neuroprotective activity of the drug shown by the LDHrelease assay (Figure 2A,B).

Furthermore, we checked the immunoreactivity to c-casp-3 in neurons exposed to CSP-1103(0.5–3 µM) for 24 h in the post-OGD period. Cells were processed for immunocytochemistry, usingan antibody specific for c-casp-3 and counterstained with hematoxylin. The data showed increasedimmunoreactivity for c-casp-3 in cells exposed to OGD. The immunoreactivity was reduced in neuronstreated with CSP-1103 at concentrations ≥0.5 µM (Figure 2C–H).

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Figure 2. Effect of CSP-1103 and ibuprofen on caspase-3 cleavage in cortical neurons exposed to OGD. (A) Representative images and (B) densitometry analysis of western blot (WB) for c-casp-3 in cytosolic extracts of cells exposed to OGD with or without drugs during 6 h recovery. CSP-1103, but not ibuprofen, was able to reduce c-casp-3 to the basal level. Bars (mean ± SEM) represent the percentage of the casp-3/actin ratio, relative to the OGD value. (C–G) Representative images of immunocytochemistry for c-casp-3 and (H) percentages of c-casp-3-positive cells counted after 24 h recovery ((C) vehicle; (D) OGD; (E) CSP-1103 0.5 μM; (F) CSP-1103 1 μM; and (G) CSP-1103 3 μM). CSP-1103 reduced the number of c-casp-3 immunopositive cells. Bars (mean ± SEM) represent the percentage of c-casp3-positive neurons compared to the total cell number. * p < 0.05, *** p < 0.001 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

2.3. CSP-1103, but Not Ibuprofen, Prevents Cytochrome C Release Induced by OGD in Primary Cortical Neurons

As a further marker of apoptosis, we investigated the levels of cytochrome c released from mitochondria after OGD exposure. This mitochondrial protein is an early signal of apoptosis that can activate the intrinsic and the extrinsic apoptotic pathways, both of which converge to activate the effector enzyme caspase-3 [24–26]. In line with previous evidence [22], the level of cytosolic cytochrome c was already detectable 2 h after OGD and further increased at 6 h. CSP-1103 significantly prevented cytochrome-c release from 2 h on (Figure 3A–D).

Ibuprofen did not modify the cytochrome release at any time. These results suggest that, conversely to CSP-1103, ibuprofen cannot limit either apoptosis or secondary necrosis in pure neuronal cultures exposed to OGD. The finding is also consistent with previous evidence showing that ibuprofen-induced neuroprotection in brain ischemia is strictly mediated by the reduction of glial reactivity and is not reproduced in pure neuronal cells [27,28].

Figure 2. Effect of CSP-1103 and ibuprofen on caspase-3 cleavage in cortical neurons exposed toOGD. (A) Representative images and (B) densitometry analysis of western blot (WB) for c-casp-3in cytosolic extracts of cells exposed to OGD with or without drugs during 6 h recovery. CSP-1103,but not ibuprofen, was able to reduce c-casp-3 to the basal level. Bars (mean ± SEM) represent thepercentage of the casp-3/actin ratio, relative to the OGD value. (C–G) Representative images ofimmunocytochemistry for c-casp-3 and (H) percentages of c-casp-3-positive cells counted after 24 hrecovery ((C) vehicle; (D) OGD; (E) CSP-1103 0.5 µM; (F) CSP-1103 1 µM; and (G) CSP-1103 3 µM).CSP-1103 reduced the number of c-casp-3 immunopositive cells. Bars (mean ± SEM) represent thepercentage of c-casp3-positive neurons compared to the total cell number. * p < 0.05, *** p < 0.001 vs.OGD value. +, presence of OGD; −, absence of OGD or treatment.

2.3. CSP-1103, but Not Ibuprofen, Prevents Cytochrome C Release Induced by OGD in Primary Cortical Neurons

As a further marker of apoptosis, we investigated the levels of cytochrome c released frommitochondria after OGD exposure. This mitochondrial protein is an early signal of apoptosis thatcan activate the intrinsic and the extrinsic apoptotic pathways, both of which converge to activatethe effector enzyme caspase-3 [24–26]. In line with previous evidence [22], the level of cytosoliccytochrome-c was already detectable 2 h after OGD and further increased at 6 h. CSP-1103 significantlyprevented cytochrome-c release from 2 h on (Figure 3A–D).

Ibuprofen did not modify the cytochrome release at any time. These results suggest that,conversely to CSP-1103, ibuprofen cannot limit either apoptosis or secondary necrosis in pureneuronal cultures exposed to OGD. The finding is also consistent with previous evidence showingthat ibuprofen-induced neuroprotection in brain ischemia is strictly mediated by the reduction of glialreactivity and is not reproduced in pure neuronal cells [27,28].

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Figure 3. Effect of CSP-1103 and ibuprofen on cytochrome c in cortical neurons exposed to OGD. (A) Representative images and (B) densitometry analysis of WB for Cyt C in cytosolic extracts after 2 h of recovery. CSP-1103, but not ibuprofen, reversed the OGD-induced cytochrome c release from mitochondria after 2 h of recovery. Bars (mean ± SEM) represent the percentage of the Cyt C/actin ratio relative to the OGD value. (C) Representative images and (D) densitometry analysis of WB for Cyt C in cytosolic extracts after 6 h of recovery. The pro-apoptotic release of cytochrome c was blocked by CSP-1103 treatment of neurons after 6 h of recovery. Bars (mean ± SEM) represent the percentage of the Cyt C/actin ratio, relative to the OGD value. *** p < 0.001 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

2.4. Effect of CSP-1103 and Ibuprofen, on p38 MAPK, GSK-3β, and NF-κB Activation in Primary Cortical Neurons Exposed to OGD

Inhibition of p38 mitogen-activated protein kinase (MAPK) activity has been shown to provide neuroprotection in cerebral ischemia [29]. To evaluate the activation state of the p38 MAPK pathway, the ratio between the densitometry value of the activated p38 form (p-p38) and total p38 was calculated. A WB analysis, using antibodies against either the active (p-Thr180/Tyr182) p38 form or total p38, was performed in the cytoplasmic extracts of cells exposed to OGD and 2 h of recovery. In line with previous evidence, no increase in p-p38 was detected in the cortical neurons exposed to OGD [30]. However, 2 h of treatment with CSP-1103 in the post-OGD period strongly reduced the p38 phosphorylation to a level below the basal value (Figure 4A,B). The treatment with ibuprofen did not modify the p38 activation state (Figure 4A,B).

Figure 3. Effect of CSP-1103 and ibuprofen on cytochrome c in cortical neurons exposed to OGD.(A) Representative images and (B) densitometry analysis of WB for Cyt C in cytosolic extracts after2 h of recovery. CSP-1103, but not ibuprofen, reversed the OGD-induced cytochrome c release frommitochondria after 2 h of recovery. Bars (mean ± SEM) represent the percentage of the Cyt C/actinratio relative to the OGD value. (C) Representative images and (D) densitometry analysis of WB forCyt C in cytosolic extracts after 6 h of recovery. The pro-apoptotic release of cytochrome c was blockedby CSP-1103 treatment of neurons after 6 h of recovery. Bars (mean ± SEM) represent the percentageof the Cyt C/actin ratio, relative to the OGD value. *** p < 0.001 vs. OGD value. +, presence of OGD;−, absence of OGD or treatment.

2.4. Effect of CSP-1103 and Ibuprofen, on p38 MAPK, GSK-3β, and NF-κB Activation in Primary CorticalNeurons Exposed to OGD

Inhibition of p38 mitogen-activated protein kinase (MAPK) activity has been shown to provideneuroprotection in cerebral ischemia [29]. To evaluate the activation state of the p38 MAPK pathway,the ratio between the densitometry value of the activated p38 form (p-p38) and total p38 was calculated.A WB analysis, using antibodies against either the active (p-Thr180/Tyr182) p38 form or total p38, wasperformed in the cytoplasmic extracts of cells exposed to OGD and 2 h of recovery. In line with previousevidence, no increase in p-p38 was detected in the cortical neurons exposed to OGD [30]. However,2 h of treatment with CSP-1103 in the post-OGD period strongly reduced the p38 phosphorylationto a level below the basal value (Figure 4A,B). The treatment with ibuprofen did not modify the p38activation state (Figure 4A,B).

A signaling molecule also involved in the pathogenesis of post-ischemic brain injury is theglycogen synthase kinase-3β (GSK-3β) [31,32]. Since chronic treatment with CSP-1103 was found toreduce the brain level of GSK-3β and concomitantly increase the kinase phosphorylated inactive formin a mouse model of AD [33], we investigated one possible rapid activation of the kinase in neuronalcultures exposed to OGD. The immunoblot analysis of either the inactive (pSer9) GSK-3β or totalGSK-3β was performed in cell extracts 6 h after the OGD. The p-GSK-3β/GSK-3β ratio showed onlya trend to decrease 6 h after the OGD exposure, consistent with a minor, or just initial, activation ofGSK-3β at that time [34]. The trend was reverted by CSP-1103, but not by ibuprofen (Figure 4C,D).

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No differences were found in the total GSK-3β level both in vehicle and treated neurons exposed toOGD (Figure S2).Int. J. Mol. Sci. 2017, 18, 184 6 of 13

Figure 4. Effect of CSP-1103 and ibuprofen on p38 mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3β (GSK-3β) in cortical neurons exposed to OGD. (A) Representative WB images and (B) densitometric analysis of activated p38 form (phospho-p38) and total p38 in the cytosolic extracts of neuronal cells after OGD and 2 h of recovery. CSP-1103, but not ibuprofen, reduced the phospho-p38/p38 ratio. Bars (mean ± SEM) represent the percentage of the phospho-p38/p38 ratio, relative to the OGD value; (C) Representative WB images of the inactivated GSK-3β form (phospho-GSK-3β) and the total GSK-3β in the cytosolic extracts of neuronal cells after 6 h of recovery; (D) Densitometric analysis represents the ratio between phospho-GSK-3β and total GSK-3β. CSP-1103, but not ibuprofen, reverted the trend to a decrease of the phospho-GSK-3β/GSK-3β ratio after OGD and 6 h of recovery. Bars (mean ± SEM) represent the percentage of the phospho-GSK-3β/GSK-3β ratio, relative to the OGD value. * p < 0.05 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

A signaling molecule also involved in the pathogenesis of post-ischemic brain injury is the glycogen synthase kinase-3β (GSK-3β) [31,32]. Since chronic treatment with CSP-1103 was found to reduce the brain level of GSK-3β and concomitantly increase the kinase phosphorylated inactive form in a mouse model of AD [33], we investigated one possible rapid activation of the kinase in neuronal cultures exposed to OGD. The immunoblot analysis of either the inactive (pSer9) GSK-3β or total GSK-3β was performed in cell extracts 6 h after the OGD. The p-GSK-3β/GSK-3β ratio showed only a trend to decrease 6 h after the OGD exposure, consistent with a minor, or just initial, activation of GSK-3β at that time [34]. The trend was reverted by CSP-1103, but not by ibuprofen (Figure 4C,D). No differences were found in the total GSK-3β level both in vehicle and treated neurons exposed to OGD (Figure S2).

Finally, we evaluated the activation of nuclear factor κB (NF-κB), a constitutively expressed transcription factor involved in pro-apoptotic and pro-inflammatory gene expression [35]. We measured the nuclear translocation of NF-κB by WB analysis of the RelA subunit in nuclear extracts from neuronal cells exposed to the vehicle or the drugs in the 2 h post-OGD. CSP-1103, but not ibuprofen, reduced the immunoreactivity of the RelA subunit in nuclear extracts, suggesting a reduced nuclear translocation (Figure 5A,B).

Figure 4. Effect of CSP-1103 and ibuprofen on p38 mitogen-activated protein kinase (MAPK) andglycogen synthase kinase-3β (GSK-3β) in cortical neurons exposed to OGD. (A) Representative WBimages and (B) densitometric analysis of activated p38 form (phospho-p38) and total p38 in thecytosolic extracts of neuronal cells after OGD and 2 h of recovery. CSP-1103, but not ibuprofen, reducedthe phospho-p38/p38 ratio. Bars (mean ± SEM) represent the percentage of the phospho-p38/p38ratio, relative to the OGD value; (C) Representative WB images of the inactivated GSK-3β form(phospho-GSK-3β) and the total GSK-3β in the cytosolic extracts of neuronal cells after 6 h of recovery;(D) Densitometric analysis represents the ratio between phospho-GSK-3β and total GSK-3β. CSP-1103,but not ibuprofen, reverted the trend to a decrease of the phospho-GSK-3β/GSK-3β ratio after OGDand 6 h of recovery. Bars (mean ± SEM) represent the percentage of the phospho-GSK-3β/GSK-3βratio, relative to the OGD value. * p < 0.05 vs. OGD value. +, presence of OGD; −, absence of OGDor treatment.

Finally, we evaluated the activation of nuclear factor κB (NF-κB), a constitutively expressedtranscription factor involved in pro-apoptotic and pro-inflammatory gene expression [35].We measured the nuclear translocation of NF-κB by WB analysis of the RelA subunit in nuclearextracts from neuronal cells exposed to the vehicle or the drugs in the 2 h post-OGD. CSP-1103, but notibuprofen, reduced the immunoreactivity of the RelA subunit in nuclear extracts, suggesting a reducednuclear translocation (Figure 5A,B).

This result was supported by analysis of the DNA binding activity of RelA, measured byDNA-based enzyme-linked immunosorbent assay (ELISA) in nuclear extracts. When comparedto the vehicle condition, treatment with CSP-1103, but not ibuprofen, reduced the NF-κB RelA bindingactivity (Figure 5C).

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Figure 5. Effect of CSP-1103 and ibuprofen on nuclear levels of RelA in cortical neurons exposed to OGD. (A) Representative images and (B) densitometric analysis of WB for RelA in nuclear extracts after 2 h recovery. When added after the OGD, only CSP-1103 was able to revert the nuclear increase of RelA. Bars (mean ± SEM) represent the percentage of the RelA/actin ratio, relative to the OGD value; (C) ELISA analysis of the DNA binding activity of RelA in nuclear extracts prepared 2 h after the OGD. CSP-1103, but not ibuprofen, reduced the DNA binding activity of RelA. Bars (mean ± SEM) represent the percentage of the DNA binding activity over the basal value, relative to OGD. * p < 0.05, *** p < 0.001 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

This result was supported by analysis of the DNA binding activity of RelA, measured by DNA-based enzyme-linked immunosorbent assay (ELISA) in nuclear extracts. When compared to the vehicle condition, treatment with CSP-1103, but not ibuprofen, reduced the NF-κB RelA binding activity (Figure 5C).

3. Discussion

Since previous studies have shown that CSP-1103 “prevents” the AICD-mediated pro-apoptotic transcription in AD mice [18] and the apoptotic cascade in cultured neurons [20,21], we here explored the neuroprotective potential of CSP-1103 using a post-ischemic paradigm. The effect of CSP-1103 was compared to that of ibuprofen, which has previously been proposed as a neuroprotective agent.

We investigate the capability of CSP-1103 and ibuprofen to interfere with the pro-apoptotic pathways activated in primary cultures of cortical neurons after exposure to OGD. This cell-based model of brain ischemia provides insights into cellular mechanisms of post-ischemic injury and drug activities that have been widely validated by in vivo studies [22,23]. The neuroprotection elicited by CSP-1103, added in the post OGD period, comprised the inhibition of the cytochrome c- and caspase-3 dependent apoptotic cascade, as well as inhibition of necrosis and the p38 and NF-κB signaling pathways.

CSP-1103 reduced the activation of caspase-3 as well as the cytoplasmic release of cytochrome c from mitochondria, which are early markers of apoptotic pathway activation. In addition, cortical neurons exposed to 3 h of OGD and a subsequent 24 h of recovery displayed an increased release of

Figure 5. Effect of CSP-1103 and ibuprofen on nuclear levels of RelA in cortical neurons exposed toOGD. (A) Representative images and (B) densitometric analysis of WB for RelA in nuclear extractsafter 2 h recovery. When added after the OGD, only CSP-1103 was able to revert the nuclear increaseof RelA. Bars (mean ± SEM) represent the percentage of the RelA/actin ratio, relative to the OGDvalue; (C) ELISA analysis of the DNA binding activity of RelA in nuclear extracts prepared 2 h afterthe OGD. CSP-1103, but not ibuprofen, reduced the DNA binding activity of RelA. Bars (mean ± SEM)represent the percentage of the DNA binding activity over the basal value, relative to OGD. * p < 0.05,*** p < 0.001 vs. OGD value. +, presence of OGD; −, absence of OGD or treatment.

3. Discussion

Since previous studies have shown that CSP-1103 “prevents” the AICD-mediated pro-apoptotictranscription in AD mice [18] and the apoptotic cascade in cultured neurons [20,21], we here exploredthe neuroprotective potential of CSP-1103 using a post-ischemic paradigm. The effect of CSP-1103 wascompared to that of ibuprofen, which has previously been proposed as a neuroprotective agent.

We investigate the capability of CSP-1103 and ibuprofen to interfere with the pro-apoptoticpathways activated in primary cultures of cortical neurons after exposure to OGD. This cell-basedmodel of brain ischemia provides insights into cellular mechanisms of post-ischemic injury and drugactivities that have been widely validated by in vivo studies [22,23]. The neuroprotection elicitedby CSP-1103, added in the post OGD period, comprised the inhibition of the cytochrome c- andcaspase-3 dependent apoptotic cascade, as well as inhibition of necrosis and the p38 and NF-κBsignaling pathways.

CSP-1103 reduced the activation of caspase-3 as well as the cytoplasmic release of cytochromec from mitochondria, which are early markers of apoptotic pathway activation. In addition, corticalneurons exposed to 3 h of OGD and a subsequent 24 h of recovery displayed an increased releaseof the enzyme LDH, a correlate of late necrotic neuronal death [22,23]. Treatment with CSP-1103 inthe recovery period protected neuronal cells from death, as demonstrated by the decrease of LDHin the culture medium. These results are in line with previous studies showing the anti-apoptoticactivity of CSP-1103 both in hippocampal neurons exposed to OGD [21] and in the SH-SY5Y cell linetreated with Aβ25-35 or TRAIL [20]. Though, while the previous studies investigated the efficacy

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of CSP-1103 “pretreatment”, the present data provide evidence of the ability of CSP-1103 to limitthe apoptotic cascade when added in the post-injury period, a condition important from a potentialtherapeutic standpoint.

Both p38 MAPK and NF-κB signaling pathways showed a direct involvement in the pathogenesisof brain ischemia [22,29,34,35], also by stimulating the production of pro-inflammatory cytokines [36–38].In cortical neurons, CSP-1103 inhibited the p38 MAPK and NF-κB pathways activation within 2 hafter OGD. Conversely, only a minor GSK-3β activation was evident within 6 h after the OGD. At thattime point, CSP-1103 was able to reverse the trend without significantly affecting either the inhibitoryphosphorylation of GSK-3β or the total GSK-3β content. This result is in line with the unchanged levelsof total p-GSK-3β detected in pure cortical neurons exposed for 18 h to CSP-1103 or ibuprofen, in spiteof the increased p-GSK-3β/GSK-3β ratio observed in AD mice treated for 6 consecutive months witheither drug [33].

Our investigations show that the anti-apoptotic neuroprotective effect of CSP-1103 is not sharedby ibuprofen. Ibuprofen showed no direct neuroprotective activity in primary neurons, though whenadministered in animal models of global ischemia, it was found to decrease neuronal damage, increasecerebral blood flow and ameliorate neurological outcome [28,39–41]. In animal models of focal ischemia,ibuprofen reduced infarct size [42,43]. Further studies in mixed cell cultures and brain slices exposedto glutamatergic excitotoxicity or to OGD [27,28,44] established that ibuprofen could limit neuronalcell death, though its activity was strictly dependent on the presence of glial cells [27]. The presentstudy confirms that evidence by showing a lack of interaction with the pro-apoptotic pathway byibuprofen and a lack of neuroprotection in pure cortical neurons exposed to OGD.

This paper supports multiple lines of evidence showing that the structural modifications designedto eliminate COX inhibitory activity in CSP-1103 produced a compound with unique pharmacologicalproperties. It is reasonable to hypothesize that AICD, a molecular target of CSP-1103, could contributeto the apoptotic pathway and inflammatory process triggered by brain ischemia. While severalevidences showed increased APP processing in cerebral regions affected by brain ischemia [45,46],so far the precise role of AICD in stroke pathophysiology and signaling cascades remains almostunexplored. AICD has been identified as a positive regulator of apoptosis because of its transcriptionalactivation of pro-apoptotic KAI1, p53, and GSK-3β genes [47]. Moreover AICD was found to directlybind and activate cytoplasmic GSK-3β in mouse models overexpressing AICD [48,49]. In the presentstudy, we detected only a non-significant increase in GSK-3β activation at the early time point weconsidered (6 h after OGD). Although these data seem to exclude the GSK-3β inhibition in the rapidanti-apoptotic effect of CSP-1103, a later involvement of this pathway cannot be excluded. Definitely,whether and how AICD generation affects brain ischemia and is implicated in the neuroprotectiveactivity of CSP-1103 deserves further investigation.

4. Materials and Methods

4.1. Oxygen-Glucose Deprivation in Nearly Pure Primary Mouse Cortical Neurons

Primary mouse cortical neurons were prepared as previously described [50]. All animal studieswere approved by the Animal Research Committees of the University of Brescia and follow theDirective 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on theprotection of animals used for scientific purposes. Briefly, C57BL/6J mice were purchased fromCharles River, Lecco, Italy. Fifteen-day embryonic mice were harvested with caesarean section fromanaesthetized pregnant dams. Cerebral cortices were isolated and dissociated by manual dispersionwith a fire-polished Pasteur pipette. The cells were plated in Neurobasal medium supplemented with2% B27, 0.5 mM L-glutamine, and 50 U/mL penicillin/streptomicin. At 11 days in vitro, neurons wereincubated with warm deoxygenated glucose-free balanced salt solution (BSS: KCl 5.36 mM, NaCl116.35 mM, MgSO4 0.81 mM, and NaH2PO4 1.01 mM), transferred to an air-tight chamber, fluxed withan anaerobic gas mixture (95% N2 and 5% CO2) to remove oxygen, and then incubated at 37 ◦C for 3 h.

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At the end of OGD, cortical neurons were allowed to recover in Neurobasal medium containing 0.4%B27 supplement under normoxic conditions and with CSP-1103 (formerly CHF5074) or ibuprofen in0.2% DMSO or with a vehicle. Cell death was evaluated after 24 h recovery. Protein extraction wasperformed 2 or 6 h after the OGD. Neuronal cell death by necrosis was evaluated using the CytoTox96-non-radioactive cytotoxicity assay (Promega, Madison, WI, USA). LDH release was calculated asthe amount of LDH released into the culture medium relative to the total releasable LDH, obtained byincubating the cells for 30 min with 1% Triton X-100. Values are expressed as a percentage of LDHreleased by cells exposed to OGD. The neuronal apoptosis was evaluated by measuring the level ofcytochrome c (WB) and cleaved-caspase-3 (immunocytochemistry and WB) at the indicated times.

4.2. Immunocytochemistry

After exposure to 3 h of OGD and 24 h of recovery, primary cortical neurons were fixed for 15 minwith Immunofix (Bio-Optica, Milan, Italy). Cells were incubated for 15 min with 0.2% Igepal and 0.3%H2O2 in 0.1 M PBS to inhibit endogenous peroxidases, then blocked for 1 h in 0.1 M phosphate-bufferedsaline (PBS) containing 3% bovine serum albumin (BSA) and 0.2% Igepal. Neurons were incubated for2 h at 37 ◦C with rabbit polyclonal anti-cleaved caspase-3 antibody (1:800, #AF835 R&D, Minneapolis,MN, USA) in 0.1 M PBS containing 3% BSA and 0.2% Igepal. The primary antibody was detectedby biotinylated anti-rabbit secondary antibody (1:600, Vector Laboratories, Burlingame, CA, USA)in PBS 0.1 M and 1% BSA, incubated for 1 h in the dark. The signal was revealed by incubationfor 45 min in the dark with ABComplex (Vector Laboratories, Burlingame, CA, USA), visualizedwith 3,3′-Diaminobenzidine (DAB) (Sigma Aldrich, St. Louis, MO, USA) and 1% H2O2 in 0.1 M PBS.The cells were subsequently counter-stained with hematoxylin, dehydrated in ethanol, and mountedwith DPX upon slides. For all procedures except the final DAB reaction, PBS was used as a washingbuffer. Quantification of cell apoptosis was performed by counting c-caspase-3 positive cells andhematoxylin stained neurons and data were expressed as percentages of c-casp-3-positive cells to totalcell number.

4.3. Western Blot Analysis

Analyses of pro-apoptotic proteins in cytosolic extracts were performed as previouslydescribed [22,51]. Briefly, cells were resuspended in 100 µL of lysis buffer (KH2PO4 1.06 mM, NaCl155.17 mM, Na2HPO4·7H2O 2.96 mM, KCl 80 mM, sucrose 250 mM, AEBSF 1 mM, aprotinin 10 µg/mL,pepstatin 1 µM, and digitonin 0.1 mg/mL, pH 7.4). They were incubated on ice for 15 min andcentrifuged at 15,000× g (15 min, 4 ◦C). The protein lysates (20 µg/sample) were processed for WBanalysis using the following primary antibodies; polyclonal anti-caspase-3 antibody (1:500, #9662 CellSignaling, Danvers, MA, USA), monoclonal anti-cytochrome c antibody (1:300, sc13156 Santa CruzBiotechnology, Dallas, TX, USA), polyclonal anti-p38 MAPK (1:500, #9212 Cell Signaling, Danvers, MA,USA), monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (1:500, #4511 Cell Signaling,Danvers, MA, USA), and polyclonal anti-actin antibody (1:1000, #A5060 Sigma Aldrich, St. Louis,MO, USA).

In order to analyze RelA activation, nuclear extracts were prepared as previously described [50,52].Briefly, cells were scraped in 400 µL cold Buffer A (10 mM HEPES-KOH pH 7.9 at 4 ◦C, 1.5 mM MgCl2,10 mM KC1, 0.5 mM dithiothreitol, and 0.2 mM phenylmethanesulfonyl fluoride) and resuspended byflicking the tube. Cells were incubated on ice for 10 min and then centrifuged (10 s). The pellet wasprocessed for high-salt extraction by resuspension in cold Buffer C (20 mM HEPES-KOH pH 7.9, 25%glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM PMSF) andincubation on ice for 20 min. Cellular debris was removed by centrifugation for 2 min at 4 ◦C and thesupernatant fraction (containing nuclear proteins) was then stored at−80 ◦C. For WB analyses, nuclearextracts (25 µg protein/sample) were resolved by 4%–12% SDS/polyacrylamide gel. Immunodetectionwas performed by incubating the membrane overnight at 4 ◦C with the primary antibody, polyclonalanti RelA antibody (1:200, sc-372, Santa Cruz Biotechnology, Dallas, TX, USA).

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The immunoreaction was revealed by 1 h incubation at 37 ◦C with secondary antibodiescoupled to horseradish peroxidase (HRP) (1:5000, NA934 GE Healthcare, Chicago, IL, USA) andchemiluminescence detection using enhanced chemiluminescence (ECL) western blotting reagents(RPN2132, GE Healthcare, Chicago, IL, USA). Quantification of immunoblots was performed bydensitometric scanning of the exposed film using Gel Pro.3 analysis software (MediaCybernetics,Rockville, MD, USA).

4.4. DNA-Based ELISA

Binding of mouse RelA to the NF-κB binding consensus sequence was evaluated by theELISA-based Trans-Am NF-κB kit (Active Motif, Carlsbad, CA, USA). The analysis procedure wasperformed as recommended by the manufacturer. 30 µg of nuclear extracts were transferred to 96-wellplates containing high density immobilized κB oligonucleotides. The active form of the RelA subunitin whole-cell extracts was detected using a specific antibody for the subunit, bound to the target DNA.Incubation with the primary antibody was followed by incubation with the HRP-conjugated secondaryantibody. After the addition of developing solutions, the samples were read by spectrophotometry.Data are analyzed by subtracting the absorbance value observed in the presence of nuclear proteinsfrom that obtained in the absence of nuclear proteins.

4.5. Statistical Analysis

All results were expressed as mean± SEM (standard error of the mean). Data were analyzed withone-way analysis of variance (ANOVA), followed by Dunnet’s multiple comparison test. p < 0.05 wasconsidered significant.

5. Conclusions

In conclusion, this study reveals the ability of CSP-1103 to prevent neuronal death and apoptosisin an in vitro model of post-ischemic brain injury. These data, together with pre-clinical and clinicalresults showing the capability of the compound to reduce pre-clinical and clinical markers ofneuroinflammation [13–15,17], suggest that CSP-1103 may have the potential for the treatment ofbrain ischemia and support the need of detailed studies in animal models of stroke.

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/1/184/s1.

Acknowledgments: This work was funded by Chiesi Farmaceutici, Parma, Italy, Ricerca Finalizzata Ministerodella Salute RF-2010–2315142 and by H607962–1 ITN Neuroinflammation. We thank Daniel Chain andRichard Margolin (CereSpir Inc., New York, NY, USA) for helpful discussions.

Author Contributions: Vanessa Porrini, Ilenia Sarnico, and Marina Pizzi conceived and designed the experiments;Vanessa Porrini, Ilenia Sarnico, Marina Benarese, Caterina Branca, and Annamaria Lanzillotta, performed theexperiments; Vanessa Porrini, Mariana Mota, Arianna Bellucci, Edoardo Parrella, and Lara Faggi analyzed thedata; Bruno Pietro Imbimbo contributed reagents/materials/analysis tools; Vanessa Porrini, Mariana Mota,Pierfranco Spano, and Marina Pizzi wrote the paper.

Conflicts of Interest: This study was supported in part by Chiesi Farmaceutici, Parma, Italy. Bruno Pietro Imbimbo isan employee of Chiesi Farmaceutici. The other authors declare no conflict of interest.

Abbreviations

TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelingNSAID non-steroidal anti-inflammatory drugAD Alzheimer’s diseaseAβ β-amyloidsCD40L soluble CD40 ligandTNF-α tumor necrosis factor αCSF cerebrospinal fluidAICD amyloid precursor protein intracellular domainTRAIL tumor necrosis factor related apoptosis inducing ligandOGD oxygen-glucose deprivation

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LDH lactate dehydrogenasec-casp-3 cleaved caspase-3 proteinWB western blotMAPK mitogen-activated protein kinaseGSK-3β glycogen synthase kinase-3βNF-κB nuclear factor kappa BDAB 3,3′-diaminobenzidineHRP horseradish peroxidase

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